Infectious pathogens can induce a fulminant innate response that contributes importantly to end- organ damage and to lethal sequelae such as septic shock. In 1993, we cloned murine macrophage migration inhibitory factor (MIF) as an upstream regulator of innate immunity and mediator of lethal shock. We defined MIF's ability to override glucocorticoid immunosuppression, induce sustained activation of the ERK1/2 MAP kinase, and inhibit activation-induced, monocyte/macrophage apoptosis. In the most recent funding period, we described a significant association between functional alleles in the human MIF gene (MIF) and clinical outcome from pneumonia. We also cloned the MIF receptor and identified signaling to require a complex of two transmembrane proteins: one mediating MIF-binding (CD74), and one initiating signal transduction (CD44). Our long-range goal remains to better define the role of MIF - now known to be encoded in a functionally polymorphic locus - on the pathogenesis and outcome of infection. Our overall hypothesis is that MIF plays a critical role in balancing the expression of a protective inflammatory response with one causing severe end-organ damage, and it is now supported both by experimental studies and by human genetic data. In this competitive renewal, we will follow-up on the genetic findings by pursuing the following three Specific Aims: 1. Define the Functional Impact of High- and Low-expression MIF Alleles on the Human Innate Immune Response. We will characterize the MIF-dependent responses of human monocytes and alveolar epithelial cells stimulated by TLR ligands that are representative of diverse microbial pathogens. Our working hypothesis is that human cells encoding high-expression MIF alleles will show a more activated phenotype and enhanced, downstream cytokine production when compared to cells encoding low-expression MIF alleles. 2. Establish the Utility of a Lung-specific, MIF Transgenic Mouse for the Study of Pulmonary Infection. We will study a transgenic mouse that overexpresses MIF in the lung. Such a mouse, when investigated in combination with MIF-KO mice, will enable mechanistic studies of MIF role's in the pathogenesis of different lung infections and provide an in vivo model of the responses of human subjects having high- or low-expression MIF alleles. 3. Define the Phenotype of a Tautomerase-null, MIF-Knockin Mouse in Models of Infection. We will use a genetic model to better define the structural basis for MIF action and address an unresolved question about the role of MIF's enigmatic tautomerization activity in its biologic function. We will study the responses to infection of a genetically-engineered "knockin" mouse that produces an MIF protein in which the catalytic proline that mediates tautomerase activity has been replaced by glycine (P1G-MIF). The information to be gained has important translation to human disease because it may allow for the identification of patients at risk for lethal outcome from different pathogens, whether bacteria, viruses, or emerging infectious agents. Better definition of MIF-dependent responses also will assist in the appropriate application of MIF-directed therapies, which are presently in pre-clinical development. PUBLIC HEALTH RELEVANCE: The innate response to infection has a critical role in determining whether the pathogen is eliminated or if the infected individual suffers lethal complications such as septic shock. The goal of this proposal is to understand how the cytokine, macrophage migration inhibitory factor (MIF), regulates the balance between protection from infection and severe inflammatory tissue damage. MIF is encoded in a functionally polymorphic genetic locus and recent work has shown an association between different MIF alleles and the clinical outcome from pneumonia. We hope to show how genetic variation in MIF influences the host response to particular infections, including bacterial pneumonia and influenza. If successful, this information will lead to the application of MIF-directed therapies for the treatment of particular infections and their complications. This work also will enable the identification of individuals who are at risk for severe infection and lethal outcome, so that appropriate measures can be taken to better treat their infections.